The long term goal of this project is to develop a 384-well cell invasion assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to evaluating cancer drugs for cell invasion is the lack of affordable 3-dimensional assays that are robust, reproducible, physiologically relevant, suitable for automation and cost-effective to perform. The further advancement of OrisTM technology, as described in this proposal, will form the basis of an affordable, easy to use cell-based assay capable of rapid and quantitative results that facilitates selection and evaluation of therapeutic candidates for cell invasion. The availability of a 384-well cell invasion assay that requires minimal numbers of cells and minute volumes of test compounds will accelerate drug development for cancer therapeutics. The proposed assay format will be compatible with automated liquid handling systems and high content analysis (HCA) instruments. The proposed assay will both be useful as a primary screen which can be read quickly by HTS instruments while also efficiently permitting subsequent secondary screens in the same assay wells that can be easily quantitated via HCA platforms. This is an extraordinary economic benefit that increases the knowledge-generating power of the research dollar;it conserves reagents (compounds, cells, etc) and resources (manpower) that would otherwise be consumed in repetitive testing required by other formats such as trans-membrane assays. The proposed 3D invasion assay is based on the innovative use of a biocompatible gel (BCG) centrally deposited in each well to exclude cells from adhering in the centers of the wells. As demonstrated in phase 1 activities, the BCG is an effective replacement for the Oris" silicone cell seeding stoppers used in our first generation cell motility assays. After cells are seeded, the BCG dissolves to reveal reproducible Detection Zones in the center of each well. A coating of extracellular matrix is then overlaid in the well and invasion in the x, y and z axes can begin. By eliminating the cell seeding stoppers, which both prevent access by automated liquid handling equipment and require a manual removal step to begin the assay, the BCG-based assay will offer the ability for robotic delivery of cells, media and test compounds thereby decreasing hands-on time required by laboratory personnel. In Phase 1 of this proposal, we developed a 96-well based Cell Migration Assay utilizing BCG to create a dissolving barrier that successfully formed Detection Zones in the center of assay wells. We effectively screened over 100 formulations of BCG and selected a suitable formulation that a) was compatible with both tissue culture treated and collagen I coated surfaces, b) had the ability to block cell attachment while completely dissolving in tissue culture media, c) permitted cell migration upon dissolution and d) did not interfere with cell viability or induce cytotoxicity. We developed and optimized robotic dispensing capabilities to achieve uniform and reproducible BCG deposition in a 96- well format and confirmed that BCG does not interfere with the efficacy of 4 different classes of inhibitors in this novel migration assay. The data presented from our Phase 1 studies clearly demonstrate the feasibility of a HTS-compatible cell motility assay based on accurate and precise deposition of a dissolvable, biocompatible gel that creates a temporary cell exclusion zone in 96-well assay plates. These results justify continued development of this technology for a 384-well, 3D high throughput cell invasion assay. Based on proven success in launching the Oris" cell-based assay product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The major goals of this Phase 2 proposal are to 1) miniaturize the 96-well migration assay to a 384-well invasion assay format, 2) optimize conditions for an ECM overlay;and 3) validate the assays in 3-day variability studies and dose-response titrations with well characterized inhibitors. Successful completion of these goals will provide researchers with a cost-effective 384-well 3D invasion assay that will reduce labor and materials needed for assay set-up and offer the ability to efficiently capture additional information per well by using multiplexed staining, thereby maximizing research funds and human resources.